The present invention relates to methods of gene therapy for inhibiting angiogenesis associated with tumor growth, inflammation, psoriasis, rheumatoid arthritis, hemangiomas, diabetic retinopathy, angiofibromas, and macular degeneration.
This invention also relates to animal models useful in the investigation of gene therapy-mediated inhibition of angiogenesis. The invention also relates to recombinant vectors which are useful in the disclosed gene therapy methods.
Vascular endothelial cells form a luminal non-thrombogenic monolayer throughout the vascular system. Mitogens promote embryonic vascular development, growth, repair and angiogenesis in these cells. Angiogenesis involves the proteolytic degradation of the basement membrane on which endothelial cells reside followed by the subsequent chemotactic migration and mitosis of these cells to support sustained growth of a new capillary shoot. One class of mitogens selective for vascular endothelial cells include vascular endothelial growth factor (referred to as VEGF or VEGF-A) and the homologues placenta growth factor (PlGF), VEGF-B and VEGF-C.
Human VEGF exists as a glycosylated homodimer in one of five mature processed forms containing 206, 189, 165, 145 and 121 amino acids, the most prevalent being the 165 amino acid form.
U.S. Pat. No. 5,240,848 discloses the nucleotide and amino acid sequence encoding the 189 amino acid form of human VEGF.
U.S. Pat. No. 5,332,671 discloses the nucleotide and amino acid sequence encoding the 165 amino acid form of human VEGF.
Charnock-Jones et al (1993, Biol. Reproduction 48: 1120-1128) discloses the VEGF145 splice variant m RNA.
U.S. Pat. No. 5,194,596 discloses the nucleotide and amino acid sequence encoding the 121 amino acid form of human VEGF.
The 206 amino acid and 189 amino acid forms of human VEGF each contain a highly basic 24-amino acid insert that promotes tight binding to heparin, and presumably, heparin proteoglycans on cellular surfaces and within extracellular matrices (Ferrara, et al., 1991, J. Cell. Biochem. 47: 211-218). The VEGF165 form binds heparin to a lesser extent while VEGF121 does not bind heparin.
Human PlGF is also a glycosylated homodimer which shares 46% homology with VEGF at the protein level. Differential splicing of human PlGF mRNA leads to either a 170 amino acid or 149 amino acid precursor, which are proteolytically processed to mature forms of 152 or 131 amino acids in length, respectively (Maglione, et al., 1993, Oncogene 8: 925-931; Hauser and Weich, 1993, Growth Factors 9: 259-268).
VEGF-B was recently isolated and characterized (Olofsson, et al., 1996, Proc. Natl. Acad. Sci. 93: 2576-2581; Grimmond et al., 1996, Genome Research 6: 124-131). The full length human cDNAs encode 188 and 207 amino acid precursors wherein the NH2 terminal portions are proteolytically processed to mature forms 167 and 186 amino acids in length. Human VEGF-B expression was found predominantly in heart and skeletal muscle as a disulfide-linked homodimer. However, human VEGF-B may also form a heterodimer with VEGF (id. @ 2580).
VEGF-C has also recently been isolated and characterized (Joukov, et al., 1996, EMBO J. 15: 290-298). A cDNA encoding VEGF-C was obtained from a human prostatic adenocarcinoma cell line. A 32 kDa precursor protein is proteolytically processed to generate the mature 23 kDa form, which binds the receptor tyrosine kinase, Flt-4.
VEGF-D was identified in an EST library, the full-length coding region was cloned and recognized to be most homologous to VEGF-C among the VEGF family amino acid sequences (Yamada, et al., 1997, Genomics 42:483-488). The human VEGF-D mRNA was shown to be expressed in lung and muscle.
VEGF and its homologies impart activity by binding to vascular endothelial cell plasma membrane-spanning tyrosine kinase receptors which then activates signal transduction and cellular signals. The Flt receptor family is a major tyrosine kinase receptor which binds VEGF with high affinity. At present the fit receptor family includes flt-1 (Shibuya, et al., 1990, Oncogene 5: 519-524), KDR/flk-1(Terman, et al., 1991, Oncogene 6: 1677-1683; Terman, et al., 1992, Biochem. Biophys. Res. Commun. 187: 1579-1586), and flt-4 (Pajusola, et al., 1992, Cancer Res. 52:5738-5743).
The involvement of VEGF in promoting tumor angiogenesis has spawned studies investigating possible antagonists of the process. Both polyclonal (Kondo, et al., 1993, Biochem. Biophys. Res. Commun. 194: 1234-1241) and monoclonal (Kim, et al., 1992, Growth Factors 7: 53-64; Kim, et al., 1993, Nature 362: 841-844) antibodies raised against VEGF have been shown to suppress VEGF activity in vivo. Anti-VEGF antibody strategies to interdict angiogenesis and its attendant tumor are also addressed in Kim et al. (1993, Nature 362: 841-844) and Asano et al. (1995, Cancer Research 55: 5296-5301).
Kendall and Thomas (1993, Proc. Natl. Acad. Sci. 90: 10705-10709) isolated and characterized a cDNA encoding a secreted soluble form of flt-1 from cultured human umbilical vein endothelial cells (HUVEC). The recombinant version of this protein was purified by binding to immobilized heparin. Isolated soluble flt-1 was shown to inhibit VEGF activity in vitro. No suggestion regarding gene transfer protocols were disclosed.
Millauer et al. (1994, Nature 367: 576-579) disclose in vivo inhibition of tumor angiogenesis by expression of an artificially generated flk-1 mutant in which the intracellular kinase domain but not the membrane-spanning anchor was deleted. The authors do not forward any teaching or suggestion that a soluble form of a VEGF tyrosine kinase receptor would be useful in gene therapy applications.
Neovascularization of malignant tumors is an integral process contributing to solid tumor growth and neoplastic progression (Kondo et al., 1993, Biochemical and Biophysical Research Communications 194: 1234-1241; Carrau et al., 1995, Invasion and Metastasis 15: 197-202). In this context, several studies have demonstrated a positive correlation between neovascularization in malignant tumors and poor clinical outcomes (Volm et al., 1996, Anticancer Research 16: 213-217; Toi et al., 1994, Japanese Journal of Cancer Research 85: 1045-1049; Shpitzer et al., 1996, Archives of Otolaryngologyxe2x80x94Head and Neck Surgery; 122: 865-868; Staibano et al., 1996, Human Pathology 27: 695-700; Giatromanolaki et al., 1996, J. of Pathology 179: 80-88). While the angiogenic process has several mediators, it appears that vascular endothelial growth factor (VEGF) may be a critical growth factor with respect to initiating the cascade of events stimulating new blood vessel formation in several tumor types (Toi et al., 1996, Cancer 77: 1101-1106; Maeda et al., 1996, Cancer 77: 858-63; Anan et al., 1996, Surgery 119: 333-339).
Aiello et al. (1995, Proc. Natl. Acad. Sci. USA 92:10457-10461) disclose genetically engineered chimeric extracellular VEGF receptors to block angiogenesis in non-malignant cells.
Despite recent advances in identifying genes encoding ligands and receptors involved in angiogenesis, no gene therapy application has been forwarded which overcomes the deleterious effect this process has in promoting primary tumor growth and subsequent metastasis. The present invention addresses and meets this need.
The present invention relates to methods of gene therapy for inhibiting VEGF-induced angiogenesis associated with diseases and disorders including, but not limited to, solid tumor growth, tumor metastasis, inflammation, psoriasis, rheumatoid arthritis, hemangiomas, angiofibromas, diabetic retinopathy, and macular degeneration. These disorders are related in that VEGF acts as a mitogen to stimulate local angiogenesis from vascular endothelial cells which in turns exacerbates the condition.
The present invention relates to gene transfer of a DNA vector and concomitant in vivo expression of a soluble form of a tyrosine receptor kinase (sVEGF-R) within the mammalian host which binds VEGF or a VEGF homologue in and around the localized site of the disorder. The formation of a sVEGF-RA/VEGF complex will inhibit binding of VEGF to the FLT-1 and KDR tyrosine kinase receptors spanning the vascular endothelial cell membrane, thus preventing initiation of the signal transduction stimulating angiogenesis. In addition, expression of sVEGF-R may also impart a therapeutic effect by binding to membrane associated VEGF-Rs. VEGF-Rs are thought to be dimerized by binding dimeric VEGF ligand which in turn allows the receptor intracellular tyrosine kinase domains to transphosphorylate each other generating phosphorylated tyrosine residues that facilitate the subsequent binding and activation of downstream signal transduction proteins. sVEGF-Rs can form heterodimers with full-length VEGF-Rs that, because the sVEGF-Rs are devoid of an intracellular tyrosine kinase region, prevent receptor tyrosine kinase domain transphosphorylation, the initiation of signal transduction and thus VEGF-induced mitogenesis and angiogenesis in a dominant negative manner.
A nucleotide sequence encoding a sVEGF-R for inclusion in a gene therapy vector of the present invention may be chosen from a group of genes encoding tyrosine kinase receptors, namely from the group consisting of sflt-1, flt-1, KDR (also denoted flk-1), and flt-4. The resulting DNA fragment encodes a protein or protein fragment which binds VEGF and/or KDR/flk-1 and inhibits formation of a wild-type, functional VEGF-R/VEGF complex.
A preferred application of the present invention relates to promoting inhibition of solid tumor angiogenesis and metastasis by utilizing the disclosed gene therapy methodology. In particular, methods are disclosed for inhibition of primary tumor growth and metastasis by gene transfer of a nucleotide sequence encoding sVEGF-R to a mammalian host. The transferred nucleotide sequence transcribes mRNA and expresses sVEGF-R such that sVEGF-R binds to VEGF in extracellular regions adjacent to the primary tumor and vascular endothelial cells. Formation of a sVEGF-R/VEGF complex will prevent binding of VEGF to the KDR and FLT-1 tyrosine kinase receptors, antagonizing transduction of the normal intracellular signals associated with vascular endothelial cell-induced tumor angiogenesis. In addition, expression of sFLT-1 may also impart a therapeutic effect by binding either with or without VEGFs to form non-functional heterodimers with full-length VEGF-Rs and thereby inhibiting the mitogenic and angiogenic activities of VEGFs.
In a particular embodiment of the present invention a truncated version of a soluble or transmembrane form of FLT-1 (Shibuya, et al., 1990, Oncogene 5: 519-524) is utilized in gene therapy protocols. It will be within the purview of the skilled artisan to generate a sVEGF-R or VEGF-RTMI construct expressing a truncated FLT-1 protein which binds to VEGF, a VEGF homologue and/or dimerizes with a full-length VEGF-R inhibiting its activation on the surface plasma membrane of vascular endothelial cells (FIG. 1). Such a construct may be generated by recombinant DNA techniques known in the art using a DNA fragment encoding a partial or complete amino acid sequence of a FLT receptor. Using recombinant DNA techniques, DNA molecules are constructed which encode at least a portion of the VEGF receptor capable of binding VEGF without stimulating either mitogenesis or angiogenesis. Standard recombinant DNA techniques are used such as those found in Maniatis, et al. (1982, Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).
In another embodiment of the present invention a mutated version of FLT-1 is generated which is defective in protein kinase activity, namely a FLT-1 protein mutated at or around one or more known active sites for protein kinase activity. A flt-1 construction will express the extracellular domain, transmembrane domain and the mutated portion of the intracellular domain such that the resulting protein at least substantially inhibits related intracellular protein kinase activity.
In a preferred embodiment of the present invention, a naturally expressed alternatively spliced DNA encoding a soluble form of FLT-1 (Kendall and Thomas, 1993, Proc. Natl. Acad. Sci. 90: 10705-10709; U.S. application Ser. No. 08/232,538, now U.S. Pat. No. 5,712,380 hereby incorporated by reference; described herein as sVEGF-RI or sFLT-1 and listed as SEQ ID NO:1 (nucleotide sequence) and SEQ NO ID:2 (amino acid sequence) is the template for constructing a gene therapy vector wherein either expressed sFLT-1 or a biologically active truncated form binds VEGF and inhibits complex formation, dimerization and activation of full-length VEGF-Rs, and hence, pathological angiogenesis.
The present invention relates to both viral and non-viral recombinant vectors for delivery to the target hosts. To this end, a preferred non-viral recombinant plasmid described herein is pcDNA3/sflt-1. An especially preferred recombinant plasmid of the present invention is pcDNAIAsFLT-1, as decribed in Example Section 5.
A recombinant adenovirus (Ad) system is preferred for delivery and prolonged expression within target cells proximal to a solid tumor. A particularly useful adenovirus system used in the present invention is described in Example 4.
Any sVEGF-R construct, including but in no way limited to sVEGF-RI and biologically active truncated forms, may be delivered to the mammalian host using a vector or other delivery vehicle. DNA delivery vehicles can include viral vectors such as adenoviruses, adeno-associated viruses, and retroviral vectors. See, for example: Chu et al., 1994, Gene Therapy 1: 292-299; Couture et al., 1994, Hum. Gene Therapy. 5:, 667-277; and Eiverhand et al., 1995, Gene Therapy 2:336-343. Non-viral vectors which are also suitable include naked DNA (see Example Sections 1, 2, 3, and 5), DNA-lipid complexes, for example liposome-mediated or ligand/poly-L-Lysine conjugates, such as asialoglyco-protein-mediated delivery systems. See for example: Felgner et al., 1994, J. Biol. Chem. 269:2550-2561; Derossi et al., 1995, Restor. Neurol. Neuros. 8:7-10; and Abcallah et al., 1995, Biol. Cell 85:1-7. It is preferred that local cells such as adipose tissue cells or smooth muscle cells, as well as tumor cells, be targeted for delivery and concomitant in vivo expression of the respective sVEGF-R protein to promote inhibition of tumor angiogenesis.
A recombinant Ad/sVEGF-RI is a preferred virus for targeting cells proximal to a solid tumor.
An especially preferred recombinant Ad/sVEGF-RI virus is AdHCMVsFLT-1.
Another especially preferred recombinant Ad/sVEGF-RI virus is AdHCMVI1sFLT.
Any membrane bound (mVEGF-R) construct or any FLT-1 or KDR construct encoding a protein deficient in kinase activity may be targeted primarily to vascular endothelial cells in the vicinity of tumor growth. DNA delivery vehicles described above may be utilized to target any such gene transfer construct to vascular endothelial cells of the mammalian host.
As used herein, xe2x80x9cVEGFxe2x80x9d or xe2x80x9cVEFG-Axe2x80x9d refers to vascular endothelial growth factor.
As used herein, xe2x80x9chomologue of VEGFxe2x80x9d refers to homodimers of VEGF-B, VEGF-C, VEGF-D and PlGF and any functional heterodimers formed between VEGF-A, VEGF-B, VEGF-C, VEGF-D and PlGF, including but not limited to a VEGF-A/PlGF heterodimer.
As used herein, xe2x80x9cVEGF-Bxe2x80x9d refers to vascular endothelial growth factor-B.
As used herein, xe2x80x9cVEGF-Cxe2x80x9d refers to vascular endothelial growth factor-C.
As used herein, xe2x80x9cVEGF-Dxe2x80x9d refers to vascular endothelial growth factor-D.
As used herein, xe2x80x9cKDRxe2x80x9dor xe2x80x9cFLK-1xe2x80x9d refers to kinase insert domain-containing receptor or fetal liver kinase.
As used herein, xe2x80x9cFLT-1xe2x80x9d refers to fms-like tyrosine linase receptor.
As used herein, xe2x80x9cAdxe2x80x9d refers to adenovirus.
As used herein, xe2x80x9cHUVECxe2x80x9d refers to human umbilical vein endothelial cell(s).
As used herein, the term xe2x80x9cmammalian hostxe2x80x9d refers to any mammal, including a human being.
As used herein, xe2x80x9csVEGF-Rxe2x80x9d generically refers to a soluble form of a tyrosine kinase receptor which binds to its respective vascular endothelial growth factor such as VEGF, VEGF-B, VEGF-C, VEGF-D and PlGF without stimulating receptor activation, mitogenesis of vascular endothelial cells or angiogenesis.
As used herein, xe2x80x9csVEGF-RIxe2x80x9d or xe2x80x9csFLT-1xe2x80x9d refers to the native human soluble form of sFLT, disclosed in U.S. application Ser. No. 08/232,538 and presented herein in cDNA form (comprising SEQ ID NO:1) and protein form (SEQ ID NO:2).
As used herein, xe2x80x9cVEGF-Rsxe2x80x9d refers to a human wild-type VEGF/VEGF homologue specific tyrosine kinase receptor such as FLT-1 and KDR.
As used herein, xe2x80x9cmVEGF-Rxe2x80x9d generically refers to a human wild-type VEGF/VEGF homologue specific tyrosine kinase receptor such which is membrane bound, including but not limited to FLT-1, VEGF-RTMI, KDR, and VEGF-RTMII, as shown in FIG. 1.
It is an object of the present invention to provide gene therapy methods to inhibit angiogenesis and growth of solid tumors.
It is also an object of the present invention to utilize a gene or gene fragment of sVEGF-R in gene therapy methods to inhibit angiogenesis and growth of solid tumors.
It is also an object of the present invention to utilize sVEGF-RI in gene therapy methods to inhibit angiogenesis and growth of solid tumors.
It is an object of the present invention to disclose animal models for the determination of efficacy of FLT-1-based constructions for cell delivery and in vivo expression in the mammalian host.
It is an object of the present invention to provide recombinant DNA vectors containing sVEGF-RI constructs for use in gene therapy to locally inhibit angiogenesis in a mammalian host.